Abstract Spinal cord injury (SCI), and neurodegenerative diseases, are complicated disorders with very limited therapeutic options and are often not completely restorable. Various rehabilitative, cellular and molecular therapies have been tested in animal models. Rehabilitative therapies are limited to only certain locations of the injury, while cellular therapy involves transplantation, and is limited by infections, compatibility, and cellular viability issues. Molecular therapies for SCI aim to modulate neuronal survival, neurite outgrowth, or to enhance synaptic plasticity and neurotransmission using several growth factors like brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), etc. Unfortunately, clinical trials using systemic delivery of neuronal growth factors have failed either due to the lack of efficacy or unacceptable side effects, or both. Alternatively, targeting molecules that are intrinsic to neurons that could promote axonal regeneration were examined by delivering Cyclic AMP (cAMP), but the post-injury application has not been successful in mammals although they induced axonal sprouting in cultures. Substances targeting RhoA involved in axonal growth via cytoskeleton reorganization have been used to treat SCI in animal models. One of the substances being used is C3 Exoenzyme (C3E) from Clostridium botulinum. However, effective and specific delivery to injured neurons has remained a major challenge in developing this enzyme into a drug. This proposal aims to develop a targeted delivery system specifically to neuronal cells using non-toxic botulinum neurotoxin (B-C3E) fusion protein as a therapeutic system to treat spinal cord injury. Four specific aims are proposed for the Phase 1 of this project: 1. Construction of biologically active B-C3E chimeras: This will include design of several chimeric proteins (B-C3E, B-C3Em (mutated at aspargine -41) and C3E/HCA (to use as a control)), cloning in a plasmid vector, expression in E. coli, purification using a nickel affinity column, and in vitro biological activity of ADP ribosylation of RhoA. 2. Characterization of binding, internalization of recombinant B-C3E and cytoplasmic localization of B-C3E and C3E: The experiments will include demonstration of binding, and internalization of the B-C3E chimeras into rat spinal cord neurons RSpN, an in vitro model for SCI. 3. Functional characterization of RhoA-ADP ribosylation upon B-C3E delivery: This will include the comparison of RhoA-ADP ribosylation upon delivery of various constructs. 4. Neurite outgrowth assay: This will include Rho or ROCK inhibition to test the neurite outgrowth or myelination. Cell based assays will be developed to evaluate the efficacy of B-C3E delivery, and resulting neurite outgrowth or myelination. The proposed project is simple and innovative, taking advantage of the specific delivery capabilities of botulinum neurotoxin derived delivery system for a ?C3E-Delivery module?. The endpoint of this project is to have a drug delivery system based on botulinum neurotoxin binding and translocation domain for neuronal tissues, and demonstration of the efficacy of a drug (C3E) for spinal cord injury using the selective delivery vehicle. The impact of the successful completion of this project will have positive repercussions for developing drug against many neuronal and brain disorders which need to be delivered into the central nervous system.